Application Exploration Of Cell-free DNA From Blastocyst Culture Medium In Preimplantation Genetic Testing For Aneuploidy

Preimplantation genetic testing for aneuploidy(PGT-A)is an effective approach for embryo chromosome karyotyping.This technique facilitates healthy embryo selection for the following up in vitro fertilization and embryo transfer(IVF-ET)and is recommended in the scenarios of older pregnancy,repeated implantation failure,and recurrent abortion in assisted reproduction.Currently,the clinically used biopsy materials for implantation are either blastomere cells or trophectoderm cells absorbed after embryo being punched by laser.However,sample collection process of the blastomere cells might negatively influence embryo development.Sample collection from trophectoderm cell requires more cells,which cannot reflect the condition of the endoderm,and improper operation will affect embryo viability.In addition,the entire embryo biopsy process is not conducive to the promotion of clinical application of PGT-A considering that it requires expensive instruments,professional manipulator,and complex operation.Hence,this study explores whether cell-free DNA of blastocyst culture medium could be an alternative option that could significantly minimize embryo damage and simplify detecting operation.Cell-free DNA in the culture medium may be derived from apoptotic and necrotic cells during embryo in vitro development.The DNA debris is fragmented and released into the culture medium through the zona pellucida.In this thesis,it was found that the size distribution of the DNA in the culture medium was consistent with that of cell-free DNA after extracting 200 microliter blastocyst culture medium DNA and performing pair-end sequencing.Additionally,picogram-level DNA was found in the culture medium through q PCR of 18 culture mediums.Afterwards,this study also compared the performance of four whole genome amplification kits in the detection of cell-free DNA through sequencing 4 and 5 pairs of culture mediums and their correspondent cell samples.Performance evaluation revealed that Pico PLEX WGA kit is the most suitable kit for culture medium cell-free DNA amplification.The rest 25 culture mediums and their corresponding 25 cells were amplified with the WGA kit,libraries were constructed subsequently for low-depth whole genome sequencing.The sequence data of all 34 pairs of samples were carried out chromosome karyotype analysis.The consistency,specificity and sensitivity rate between culture medium and cell was 88.24%,81.25% and 94.44%,respectively.This paper explored the potential application of blastocyst culture medium cell-free DNA in preimplantation genetic testing for aneuploidy.This technique doesn’t need cell sampling so that it can be operated simply and safely and will not damage the embryo or affect the embryonic development.It can supply genetic information of ectoderm and endoderm at the same time.Meanwhile,this method is highly accurate and non-invasive.Collectively,our study provided technical foundation and data accumulation for the subsequent technical optimization in this field and has great potential for clinical application.